Ocular complications from diabetes are a leading cause of blindness in the United States. Throughout the next few decades, the prevalence of complications stemming from Type 2 Diabetes Mellitus (T2DM) is expected to rise, increasing the economic burden of the disease. The goals of this proposal are: (1) to define the pathophysiological mechanisms that result in painful and often refractory diabetic corneal disease and; (2) to identify biomarkers for use in detecting at-risk patients prior to the onset of sight threatening ocular disease. This proposal will test three novel hypotheses: (1) That IGF-1 and insulin mediate distinct events at the plasma membrane and in the nucleus through regulation of Hybrid-R, IGF-1R and INSR. (2) That increasing levels of inflammation and oxidative stress, and a loss of trophic support from corneal nerves, results in aberrant Hybrid-R expression and function. (3) That, in addition to regulating Hybrid-R, changes in the amount of IGF-related proteins in tears represent potential biomarkers to identify patients at-risk for significant ocular pathology in T2DM. We will test these hypotheses in the following three aims: Aim 1: (A) Evaluate specific factors that control Hybrid-R formation, insulin sensitivity and GSK3 beta activity in corneal epithelial cells. (B) Identify nuclear targets for Hybrid-R, IGF-1R, and INSR. Aim 2: (A) Test the effects of IGF-1, IGF-II, insulin, and IGFBP3 on corneal epithelial cells and nerve fiber loss in the presence of inflammatory mediators and hyperglycemia-induced oxidative stress. (B) Characterize the effects of T2DM on Hybrid-R mediated gene regulation and correlate with diabetes-related changes in the corneal subbasal nerve plexus and terminal epithelial nerves in situ. Aim 3: Determine whether tear levels of IGF-1, insulin and IGFBP3 in human patients with T2DM correlate with corneal and retinal pathology, using new quantitative microvolume biochemical assays, in vivo confocal microscopy (IVCM), and clinical markers of corneal and retinal disease. These aims will be accomplished using a combination of in vitro culture models, in situ human tissue analysis, and in vivo clinical studies to investigate how the pathophysiological effects from T2DM impacts the morphology of corneal nerves, the physiology of the tear film, and the corneal epithelium. These will be the first studies to investigate a rolefor the Hybrid-R in mediating corneal disease in diabetes. These studies also propose a novel biological mechanism for impaired local insulin signaling in corneal disease. Other laboratories are focused on impaired wound healing in diabetes. Our focus is on identifying key mechanisms that contribute to corneal damage; and characterizing and validating tear-film derived biomarkers that will allow for early, interventional approaches to prevent diabetes-related vision loss.